Parasitology Research (2019) 118:2789–2800 https://doi.org/10.1007/s00436-019-06450-3

FISH PARASITOLOGY - ORIGINAL PAPER

Chromosomal study of Khawia abbottinae (: Caryophyllidea): karyotype and localization of telomeric and ribosomal sequences after fluorescence in situ hybridization ()

Martina Orosová1 & Irena Provazníková2,3 & Bing Wen Xi4 & Mikuláš Oros1

Received: 17 June 2019 /Accepted: 28 August 2019 /Published online: 4 September 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract An original cytogenetic study combining classical karyotype analysis and modern fluorescence in situ hybridization using telomeric

(TTAGGG)n and ribosomal sequences (18S rDNA) was performed in Khawia abbottinae (Cestoda, Caryophyllidea), a parasite of Chinese false gudgeon ( rivularis) from China. Analyses based on conventional Giemsa staining, DAPI, YOYO-1 dye, and silver (Ag) staining were also carried out. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n =16, n=5m +3t). Constitutive heterochromatin was mainly positioned at pericentromeric regions, and telomeric sequences

(TTAGGG)n were restricted to the end of all chromosomes. In mitotic preparations stained with Giemsa, both homologues of chromosome pair 4 showed a distinct secondary constriction. FISH with rDNA probe confirmed that this secondary constriction contains a nucleolar organizer region (NOR). The process of spermatocyte meiosis and the dynamics of nucleolus degradation in dividing cell were scrutinized. The present study and its results enhance the limited knowledge on basic karyotype characteristics and 18S rDNA clusters location in caryophyllidean tapeworms.

Keywords Cestoda . Karyotype . FISH . 18S rDNA mapping . Telomeres

Introduction one set of reproductive organs (Mackiewicz 1994). Recent studies revealed several unusual molecular and genetic phe- Tapeworms of the order Caryophyllidea represent a unique nomena (Kráľová-Hromadová et al. 2012;Brabecetal.2012; group among the “true” cestodes () in that they are Scholz et al. 2014; Hanzelová et al. 2015; Špakulová et al. monopleuroid, i.e. their body is monozoic and contains only 2019), such as the lineage-specific nuclear paralogs of mtDNA, intraindividual divergence of ribosomal internal tran- scribed spacers 2 (ITS2) along with multiple rDNA loci, cryp- Handling Editor: Julia Walochnik tic species diversity, phenotypic variability but genetic unifor- mity, and the triploid character of some species. Knowledge * Martina Orosová [email protected] on the chromosome sets of caryophyllidean tapeworms is rather scanty; thus far, 23 tapeworm species have been studied cytogenetically, and the karyotypes complements of only 14 1 Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, species have been recognized in all details (for a review, see 040 01 Košice, Slovakia Špakulová et al. 2011; Orosová and Oros, 2012). 2 š Institute of Entomology, Biology Centre CAS, Brani ovská 31, 370 The genus Khawia Hsü, 1935, the most specious genus of 05 České Budějovice, Czech Republic the order Caryophyllidea, contains eight valid species parasit- 3 Č Faculty of Science, University of South Bohemia in eské izing cyprinid with worldwide distribution, except for Budějovice, Branišovská 1760, 370 05 České Budějovice, Czech Republic the neotropical region (Scholz et al. 2011; Scholz and Oros 2017). Khawia abbottinae Xi, Oros, Wang, Scholz and Xie, 4 Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research 2013 was originally described as a specific parasite of the Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China Chinese false gudgeon, Abbottina rivularis (, 2790 Parasitol Res (2019) 118:2789–2800

Gobioninae), from the Yangtze River basin in China (Xi et al. ribosomal genes. Additionally, FISH with the telomeric probe

2013). In total, the authors reported five species of the genus (TTAGGG)n was performed to the examined karyotype for Khawia (K. abbottinae, K. japonensis, K. rossittensis, thepossiblepresenceofinterstitial telomeric sequences K. saurogobii and K. sinensis) in China. (ITS). Moreover, the present study also focused on a descrip- Within the family Lytocestidae, chromosomes have been tion of heterochromatin distribution (DAPI-banding) and the studied only sparsely and the knowledge of karyotype organi- occurrence of nucleoli during meiosis by staining with fluo- zation has been limited to 6 species of 3 genera to date (for rescent dye YOYO-1. review see Špakulová et al. 2011; Orosová and Oros 2012). The majority of previous studies were based exclusively on a description of the diploid number, with low emphasis on chro- Materials and methods mosome morphology. For the genus Khawia, karyological analysis of 4 species, namely K. japonensis (under synonym Specimens of K. iowensis, Grey 1979), K. sinensis, K saurogobii and K. rossittensis revealed a constant diploid number, 2n =16, Fifteen alive specimens of Khawia abbottinae were obtained and similar chromosome morphology (Grey 1979; from the intestine of the Chinese false gudgeon (Abbottina Petkevičiūtė 1998; Mutafova and Nedeva 1999;Orosová rivularis) from water bodies of the middle and lower reaches et al. 2010a; Orosová and Oros 2012). Repetitive DNA anal- of the Yangtze River basin (Yangtze River in Wuhan, Hubei ysis has shown only a very low variation in the arrangement of province, and Taihu Lake in Wuxi, Jiangsu province) in April heterochromatin along the chromosomes among the studied 2011 and April 2018. Tapeworms were dissected from the species. Thus, it is difficult to identify chromosome rearrange- intestine of freshly killed fish, rinsed several times in saline ments and to study chromosome evolution within this genus solution, and processed immediately for chromosome slide based on classical karyotype analysis. Ribosomal DNAs are preparations, and subsamples of specimens were fixed in valuable cytogenetic markers in plant and genome re- 96% ethanol for DNA extraction. search, as they are presented in numerous repeated units, gen- erally organized in clusters, and thus are easily visualized in Chromosome preparations the chromosomes. Fluorescent in situ hybridization (FISH) has been used for decades for physical mapping of repeated For cytogenetic analysis, whole living tapeworms were incu- sequences, such as ribosomal DNAs on mitotic and meiotic bated in 0.025% colchicine solution (in saline) for 1 h at room chromosomes in various organisms (Nguyen et al. 2010; temperature (RT). Thereafter, they were placed into a hypo- Sheng and Wang 2010; Cabral-de-Mello et al. 2011; tonic solution of 0.75 M KCl for 2 h at RT and torn gently at Bombarová et al. 2015; Solovyeva et al. 2016; Teixeira et al. the testicular body area using the insulin syringes. The torn 2017;Supiwongetal.2019). There are rarely any molecular worms were placed into freshly prepared cold fixative solution cytogenetic studies that use FISH to map the ribosomal genes (methanol to acetic acid, 3:1) with two changes, 15 min each. of caryophyllidean tapeworms, and such data are available for The fixed material was stored at − 20 °C. only four species. In the genus Khawia, a single locus bearing Spread chromosome preparations were made using the a nuclear organizer region (NOR) was detected at the homo- method according to Orosová and Špakulová (2018). logues of pair 7 in K. saurogobii and of pair 6 in K. sinensis Shortly, small pieces containing medullary parenchyma with (Orosová et al. 2010a; Orosová and Oros 2012). In both spe- testes were transferred into a drop of 60% acetic acid on a cies, FISH with the 18S rDNA probe identified major rDNA clean slide and torn into fine pieces with the help of tungsten clusters located in the pericentromeric region on the small needles. The slide was then placed on a heating plate at 45 °C, metacentric chromosome. and the drop of cell suspension was slowly drawn along the Cytogenetic studies can contribute an array of information slide until the liquid evaporated. The preparations were passed independent from morphological and molecular characteris- through an ethanol series (70, 80, and 100%, 1 min each) and tics, which are routinely used for phylogenetic analysis. The stored at − 20 °C until further use. Before each kind of stain- differences and similarities may not be apparent at the molec- ing, preparation was removed from the freezer, dehydrated in ular or morphological level. However, they could be revealed an ethanol series, and air-dried. through karyological data (Sessions 1996). The intention of the current work was to extend the limited knowledge of chro- Karyological analysis mosomes in caryophyllidean tapeworms. An in-depth chro- mosomal analysis (number, size, morphology) in a newly de- The preparations were stained with 5% solution of Giemsa scribed species of the family Lytocestidae, K. abbottinae,was (Merck, NJ, USA) in phosphate buffer (pH 6.8) for 25 min carried out for the first time. Number and location of NORs and rinsed with distilled water. Detailed analysis (length, mor- were analysed through Ag staining and FISH with 18S phology and centromeric index of chromosomes) was Parasitol Res (2019) 118:2789–2800 2791 performed in 10 best mitotic spreads (with clearly distinguish- DNA polymerase I (ThermoFisher, Waltham, MA, USA). able chromosomes) out of 112 evaluated dividing cells from 9 The reaction time was 1 h at 15 °C. worms. The chromosomes were classified following the no- The unlabeled, about 2100-bp-long 18S rDNA probe was menclature of Levan et al. (1964). prepared by PCR from the K. abbottinae genomic DNA, using two primers, 18S-WormA forward (5′-GCGAATGGCTCATT AAATCAG-3′) and 18S-WormB reverse (5′-CTTG Silver nitrate (AgNO3) staining TTACGACTTTTACTTCC-3′). The procedure described by Littlewood and Olson (2001) was used. The genomic DNA AgNO3 staining was performed according to Howell and was extracted using a DNeasy Tissue Kit (Qiagen, Hilden, Black (1980): six drops of 50% AgNO3 solution and three drops of 2% gelatine in 1% formic acid were mixed on the Germany). The probe was labelled with biotin-16-dUTP surface of chromosome slides and coverslip. The slide was (Roche Diagnostics, Mannheim, Germany) either by PCR then incubated on a heating plate at 45 °C until the purple with dNTP mix containing 0.35 mM biotin-16-dUTP or using Giemsa-colour of the chromosome preparation changed to the improved nick translation procedure (for details, see light brown. Slides were rinsed with distilled water and air- above). The time of incubation was 50 min at 15 °C in a dried. thermocycler. FISH with biotin-labelled 18S rDNA probe YOYO-1 staining For FISH the procedure described by Sahara et al. (1999)with To visualize the nucleoli, chromosome preparations were slight modifications was used. Briefly, chromosome prepara- stained with the DNA and RNA binding dye YOYO-1, tions were treated with proteinase K (20 mg/mL) in 1 × PBS (1,1′-[1,3propanediyl-bis[(dimethylimino)-3,1- for 5 min at 37 °C, washed twice in 1 × PBS for 5 min each, propanediyl]]bis[4-[(3-methyl-2(3H)- and then digested with 100 μg/mL RNase A in 2 × SSC for 1 h benzoxazolylidene)methyl]]-quinolinum tetraiodide) at 37 °C and washed twice in 2 × SSC for 5 min each. The (Molecular Probes, Eugene, OR, USA), at a concentration of slides were incubated in 5 × Denhard’s solution for 30 min at 4 nmol/L in PBS buffer containing 1% Triton X-100 (Sigma- 37 °C. After denaturation at 68 °C the chromosome were Aldrich, St. Louis, MO, USA). Finally, the preparations were hybridized with the probe cocktail containing: 10 μl; 50% mounted in antifade based on DABCO (Sigma Aldrich, St. deionized formamide, 10% dextran sulfate in 2 × SSC, ~ Louis, MO, USA). 50 ng of biotinylated 18S rDNA probe and 25 μgofsonicated salmon sperm DNA for one slide (Sigma-Aldrich, St. Louis, DAPI staining MO, USA). The probe was denatured at 90 °C for 5 min. Hybridization at 37 °C for 20 h was followed by stringent The chromosome preparation was cleaned in phosphate- washes, which included 50% formamide in 2 × SSC (3 × buffered saline (1× PBS) containing 1% Triton X-100 for 5 min, 46 °C) (Fluka, Buchs, Switzerland), 2 × SSC (5 × μ ′ 5 min at RT, stained with 0.5 g/mL DAPI (4 ,6-diamino-2- 2 min, 46 °C), 0.1 × SSC (3 × 5 min, 62 °C), and 4 × SSC phenylidole; Sigma-Aldrich) in PBS containing 1% Triton X- containing 0.1% Tween 20 (3 × 3 min, 37 °C). Hybridization 100 for 15 min, washed in 1% Kodak-PhotoFlo (Kodak Alaris signals were detected with Cy3-conjugated streptavidin Inc., Rochester, NY, USA) for 3 min at RT, and then in 1% (Jackson ImmunoRes. Labs. Inc., West Grove, PA, USA), Kodak-PhotoFlo in miliQ water for 1 min at RT. Finally, the amplified with biotinylated anti-steptavidin (Vector Labs. slides were mounted in antifade based on DABCO (Sigma- Inc., Burlingame, CA, USA), and Cy3-conjugated Aldrich; for details, see Traut et al. 1999). streptavidin. The preparations were counterstained with 0.5 μg/mL DAPI (4′,6-diamino-2-phenylidole; Sigma- Preparation of telomeric and 18S rDNA probes Aldrich) and mounted in DABCO- based antifade (Sigma- Aldrich).

Telomeric probe (TTAGGG)n was generated by means of non-template PCR as described previously (Sahara et al. FISH with tyramide signal amplification (TSA-FISH) 1999) and labelled with Cy3-dUTP using the improved nick translation procedure of Kato et al. (2006)withslightmodifi- The telomeric probe was prepared by nick translation as de- cations. The modified 20 μL reaction contained 1 μg scribed above and purified using Sephadex (Illustra Sephadex unlabelled DNA; 50 μM dATP, dCTP, and dGTP; 10 μM G-50 fine DNA grade, GE Healthcare, Chicago, IL, USA). dTTP; 20 μM labelled nucleotides; 1× nick translation buffer For TSA-FISH, we used the procedure described in Zrzavá (50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 0.005% BSA); et al. (2018). In particular, chromosome slides were treated 10 mM β-mercaptoethanol; 0.005 U DNase I and 20 U with 10 mM HCl for 10 min at 37 °C and incubated in 1% 2792 Parasitol Res (2019) 118:2789–2800 hydrogen peroxide for 30 min at RT to quench endogenous highlighted (Fig. 3a–f). One large compact nucleolus was usu- peroxidase activity. Then, the preparations were digested with ally observed in interphase nuclei using YOYO-1 staining 100 μg/mL RNase A for 1 h at 37 °C and blocked with 5× (Fig. 3a). During the first meiotic prophase, a single nucleolus Denhard’s solution for 30 min at 37 °C. Chromosomes were was regularly detected from early leptotene to pachytene nu- denatured in a probe mix containing 10–30 ng of the labelled clei, where the nucleolus was visible as a lightly stained mass telomeric probe, 50% deionized formamide, and 10% dextran on the lateral site of the bivalent (Figs. 2d and 3b–e). sulfate in 2 × SSC for 5 min at 70 °C and hybridized over- Nucleolar remnants were sometimes observed during early night. Hybridization signals were enhanced by diplotene (Figs. 2e and 5c, d) but not in late diplotene antifluorescein-HRP (horseradish peroxidase) conjugate (Figs. 2f and 3f); thus, it disappeared before reaching diakine- (PerkinElmer, Waltham, MA, USA) diluted 1:1000 and incu- sis. In diplotene nuclei, the largest bivalent showed four to five bated with tyramide solution (TSA Plus Fluorescein system, chiasmata in the majority of evaluated cells PerkinElmer) for 5–7 min. The preparations were counter- (Figs. 2e and 5c, d), while it usually had two chiasmata in stained and mounted in DABCO-based antifade containing diakinesis (Figs. 2f and 3f). 0.5 μg/mL of DAPI. FISH with telomeric probe and localization of 18S Microscopy and image processing rDNA

The stained slides were inspected under conventional light FISH with an 18S rDNA probe revealed that the 18S rDNA and fluorescence microscopes Leica DM 4000 B equipped loci were localized in the pericentromeric region of the short with colour digital camera DFC 450 C and Olympus B51 arm of the metacentric chromosome pair 4. Positive signals of equipped with DP70 CCD camera. Black-and-white fluores- ribosomal DNAwere visible at the sites of secondary constric- cent photographs were captured, pseudocoloured (light blue tions of mitotically dividing spermatogonia. In pachytene nu- for DAPI, green for YOYO-1 and red for Cy3), and processed clei of spermatocytes, the 18S rDNA-FISH probe hybridized with Adobe Photoshop, version 7.0. close to the centromere of one NOR-bivalent, as indicated by the association with DAPI highlighted centromeric hetero- chromatin (Fig. 4d–g). Some homologue chromosomes of Results meiotic bivalents showed few smaller, inconspicuous, intersti- tial blocks of heterochromatin. FISH with an 18S rDNA Basic karyotype characteristics probe, performed after YOYO-1 staining, showed in pachy- tene spermatocytes a cluster of rDNA genes co-localized with Evaluating of 112 dividing cells from nine worms showed a nucleolus bonded with the NOR bivalent (Fig. 3e). exclusively a modal diploid number 2n =16andafundamen- As a single rDNA cluster was found in the karyotype of tal number FN = 28 (Fig. 1). In meiotic pachytene and K. abbottinae, the silver (Ag nitrate) staining was applied in diplotene/diakinesis nuclei, clumps of eight bivalents (n =8) order to reveal the activity of rDNA sites in previous cell was commonly observed (Figs. 2c–f and 3d). The division. In principle, this staining indicates proteins associat- K. abbottinae karyotype consists of eight chromosome pairs, ed with active rRNA genes (Howell and Black 1980). In most five metacentric (pair number 1, 2, 3, 4, and 5), and three interphase nuclei, AgNO3 detected the presence of single ac- telocentric (pair number 6, 7, and 8) that are shown in tive nucleolus, and in the majority of meiotic prophase chro- Fig. 1c, d. The karyotype formula can be summarized as mosomes, one large compact nucleolus was regularly ob- 2n =16; n =5m +3t. The chromosomes were comparatively served from early leptotene nuclei until early diplotene large; in 10 mitotic spreads used for karyometrical analysis, (Fig. 5). This proved that the rDNA cluster represents a tran- the largest chromosome 1 measured 10.2 μm, being thus scriptionally active NOR. markedly longer than the second metacentric pair in all the FISH with the sequence (TTAGGG)n as a DNA probe re- evaluated chromosome complements (for karyometrical vealed typical twin fluorescent signals at the ends of all chro- measurements, see Table 1). Gradual length decrease of the mosomes of K. abbottinae (Fig. 4i, j). Intensive signals were remaining seven pairs was observed. The mean total comple- discovered only at the ends of all chromatids in all analysed ment length (TLC) was 56.8 μm. At the short arm of both chromosomes. It is common that the size of the telomeric homologues of metacentric chromosome pair 4, a noticeable repeat units in the interstitial region of chromosomes is small- secondary constriction was observed in Giemsa-stained slides er than their size in the telomeric region. We therefore per- (see the inset in Fig. 1), pointing out the presence of a NOR. formed telomeric TSA-FISH on meiotic chromosomes, which The processes of meiotic division of spermatocytes were could reveal interstitial telomeric sequences (ITS). However, documented by Giemsa staining (Fig. 2) and using fluorescent no FISH signals were found in the internal chromosome YOYO-1 staining, which also allowed a nucleolus to be regions. Parasitol Res (2019) 118:2789–2800 2793

Fig. 1 Giemsa-stained chromosomes of Khawia abbottinae. a, b Mitotic metaphase, 2n =16.c, d Karyotypes derived from mitotic metaphases on a and b.Theinset in the upper right corner shows a detail of chromosomes no. 4 with distinct secondary constriction. Scale bar = 10 μm

Discussion interspecific differences in chromosome morphology expressed by the centromere position of individual pairs were In this paper, the chromosomes of the tapeworm K. abbottinae observed. As shown in Fig. 6, K. saurogobii and K. sinensis, were described for the first time. Our karyotype analysis re- both with n =3m +5t, are evidently dissimilar in the centro- vealed that K. abbottinae is a diploid species with 2n =16 mere position of the sixth and eighth chromosome pairs. chromosomes, as for the other 4 species of the genus Similarly, K. japonensis and K. abbottinae,characterizedby Khawia describedsofar:K. japonensis (Grey 1979), n =5m +3t, clearly differ from each other in relation to the K. rossittensis (Grey 1979), K. saurogobii (Orosová et al. centromere position of chromosome pairs 3, 4, 7, and 8. The 2010a)andK. sinensis (Petkevičiūtė 1998;Mutafovaand differences in chromosome morphology without variation on Nedeva 1999; Orosová and Oros 2012). However, among the chromosome number suggest the occurrence of these species, differences in chromosomal morphology are intrachromosomal changes, i.e. pericentric inversions, in the observed. Karyotypes of K. saurogobii and K. sinensis consist speciation processes of this lineage. Possible causes of the of three pairs of metacentric and five pairs of telocentric slight differences in the measurements of chromosomes were (acrocentric) chromosomes (n =3m +5t) while that of well discussed in Orosová and Oros (2012). K. japonensis and K. abbottinae contain five pairs of meta- Comparison of two nuclear ribosomal (ssrDNA and D1-D3 centric and three pairs of telocentric chromosomes (n =5m + lsrDNA) and two mitochondrial (nad3 and cox1) genes con- 3t)(Orosováetal.2010a, this paper). Identical diploid number firmed the paraphyletic status of the genus Khawia and sug- (2n = 16) and only two types of chromosomes shape (meta- gested that this genus is splitting into three major groups char- centric and telocentric) indicate the relatively high similarity acterized by their fish definitive hosts (Schloz et al. 2011; Xi of the macro-chromosomal structure in the five species of the et al. 2013). The first group conformed that K. baltica was genus Khawia. Another common feature among all species of transferred to the genus Caryophyllaeus (Barčák et al. 2017). Khawia spp. is that the first chromosome pair is significantly Recent phylogenetic analysis supported the close relationship larger than the rest of the chromosomes and also is always of K. abbottinae to the more advanced K. japonensis, metacentric. Despite obvious karyotype conformity, some K. rossittensis,andK. parva (Xi et al. 2018). The species of 2794 Parasitol Res (2019) 118:2789–2800

Fig. 2 Meiotic division of spermatocytes of Khawia abbottinae stained Early anaphase II. i Two spermatids. Arrowhead in d and arrow in g with Giemsa. a Leptotene. b Late zygotene. c, d Pachytene stages with indicate a secondary constriction of the fourth chromosome pair. eight bivalents. e Early diplotene with five chiasmata in the first bivalent. Asterisks in e and f indicate the first bivalent. Scale bar = 10 μm f Diplotene/diakinesis with two chiasmata. g First meiotic metaphase. h this lineage showed more frequent bi-armed chromosome K. japonensis,andK. rossittensis, and the last one by than mono-armed ones. K. armeniaca. The congruence of the molecular data to com- The species K. saurogobii and K. sinensis, which are very parative karyological features indicates a high capability of similar in terms of molecular features but apparently different karyotype evolution being reflected in the evolutionary histo- morphologically, shared a mutual position in the phylogenetic ry of the genus and fits into the category of karyotypic tree (Scholz et al. 2011;Xietal.2018). In both species, the orthoselection, according to White (1973). The telocentric telomeric chromosome structure prevailed over the metacen- character of majority chromosomes of K. saurogobii and tric one. All the present cytogenetic features, even though they K. sinensis could be regarded as ancestral. In contrast, more are scarce, show their validity as phylogenetically informative numerous bi-armed elements of K. abbottinae and data. K. japonensis are supposedly derived. Unfortunately, to date, Based on the current phylogenetic tree topology published the karyotype of the member of the last third Khawia clade by Xi et al. (2018, see Fig. 4), three karyotypic clades can be formed by a single K. armeniaca species is unknown. recognized. The first one is formed by K. sinensis and Insufficient karyological data make it difficult to come up K. saurogobii, the second one by K. abbottinae, with a wide generalization, but we can assume that the Parasitol Res (2019) 118:2789–2800 2795

Fig. 3 Visualization of the nucleoli (N) in meiotic cells stained with that co-localize with a cluster of hybridization signal of the 18S rDNA YOYO-1. a Interphase nucleus. b, c Zygotenes. d Pachytene. e probe and secondary spermatocyte (left). f Diplotene/diakinesis stage. Pachytene complement (right) showing one YOYO-1-stained nucleolus Arrowheads indicate the nucleolus. Scale bar = 10 μm karyotype evolution within the genus Khawia is conservative, A course of meiotic spermatocyte division coincides well proceeding without any changes in the chromosome number. with usual processes known in the majority of other In many cases, cestodes are characterized by the conservatism (Grelon 2016), and spermatogenesis proceeds as usual, with in chromosomes number within the genus (for a review, see sperm being formed after two meiotic divisions. Similarly, Špakulová et al. 2011). The main mechanism that contributed nucleolus degradation seemed to be the standard and takes to the cytogenetic divergence of Khawia species with 2n =16 place before diakinesis (Pawlovski and Cande 2005). The was apparently pericentric inversions. Pericentric inversions meiotic bivalent no. 1 displayed a comparatively high number are quite common in the cytogenetic divergence of animal of chiasmata in the diplotene nuclei. Chiasmata are important species they have also been reported in many other platyhel- in gene shuffling between homologous chromosomes (John minth parasite species (Birstein 1991; Hirai et al. 2000; 1990). The majority of studied cells of K. abbottinae showed Petkevičiūtė and Bondarenko 2001; Petkevičiūtė 2003; four to five chiasmata, indicating a higher recombination rate. Bombarová et al. 2007; Stanevičiūtė et al. 2015). Chiasma formation has not yet been investigated within

Table 1 Measurements (means ± SD) and classification of Chromosome Absolute length Relative length Centromeric index Classification chromosomes of Khawia number (mean ± SD) (μm) (mean ± SD) (%) (mean ± SD) abbottinae 1 10.2 ± 1.37 18.03 ± 2.40 45.81 ± 2.76 m 2 8.8 ± 1.07 15.49 ± 1.89 45.33 ± 2.66 m 3 8.0 ± 1.27 14.24 ± 2.06 46.14 ± 1.39 m 4 7.2 ± 1.46 12.73 ± 2.56 40.60 ± 2.60 m 5 6.5 ± 1.46 11.56 ± 2.41 41.55 ± 3.11 m 6 6.2 ± 1.74 11.00 ± 2.84 0 t 7 5.3 ± 1.54 9.23 ± 2.71 0 t 8 4.6 ± 1.37 8.10 ± 2.41 0 t

m metacentric chromosome, t telocentric chromosome 2796 Parasitol Res (2019) 118:2789–2800

Fig. 4 Chromosomes of Khawia abbottinae stained with DAPI (blue) eight bivalents. Asterisk indicates the fourth bivalent with rDNA cluster. f and 18S rDNA (a–g) and (TTAGGG)n (h–j) fluorescence in situ Early diplotene. g Spermatids with 18S rDNA signals associated with hybridization probes (red). a Interphase nucleus with a large nucleolus DAPI-highlighted block. h Interphase nucleus with hybridization and one 18S rDNA cluster. b Mitotic prometaphase. c Zygotene. d signals of telomeric probe. i Pachytene and j diplotene with Pachytene bivalents in a bouquet stage showing a cluster of interstitial hybridization signals of telomeric probe at the chromosomal ends of each rDNA signals associated with DAPI block. e Pachytene complement of bivalent. Scale bar = 10 μm monozoic tapeworms (Caryophyllidea). There are only a few Bombarová et al. 2015). Alternatively, the presence of hetero- data available; some may be evaluated from previously pub- chromatin blocks out of the centromeric region has been re- lished images of meiotic division in tapeworms (Orosová et al. ported in the caryophyllidean cestodes Caryophyllaeides 2010b; Bombarová and Špakulová 2015). It is apparent that fennica (Orosová et al. 2010b)andAtractolytocestus chiasma distribution within bivalents appears to be deter- huronensis (Kráľová-Hromadová et al. 2010; Špakulová mined by morphological features of the chromosomes. The et al. 2019). The more or less non-variable pattern of hetero- same correlation between the number of chiasmata and chro- chromatin dispersion indicates that the presence of hetero- mosome length was reported in the fluke subclass chromatin remains consistent over the evolutionary history Aspidogastrea (Petkevičiūtė 2001; Bombarová et al. 2015). within cestode lineages.

All of the Khawia species share a common feature, i. e. The telomeric repeat motif (TTAGGG)n is established as similar content of heterochromatin distribution, with conspic- the ancestral sequence for all Metazoa (Meyne et al. 1989; uous heterochromatin located in the pericentromeric region of Traut et al. 2007). All the up-to-date studied representatives almost all chromosomes. A low amount of heterochromatin as from the phylum Platyhelminthes have (TTAGGG)n telomeric well as its presence in the centromeric or pericentromeric re- repeats, and the same motif was shown to be present in three gions is a common characteristic in most of Cestoda, Digenea, tapeworm species (Bombarová et al. 2009). In addition to the Aspidogastrea, and species (Rausch and Rausch telomeric pattern at the chromosome ends, ITSs were detected 1990; Mutafova and Gergova 1994; Špakulová and Scholz in many species (Schubert 2007), and the existence of such 1999; Orosová and Oros 2012; Zadesenets et al. 2012; sequences was often related to alteration in chromosome mor- Bombarová et al. 2014; Bombarová and Špakulová 2015; phology (Meyne et al. 1990). Meiotic pachytene bivalents are Parasitol Res (2019) 118:2789–2800 2797

Fig. 5 Silver staining of meiotic cells of Khawia abbottinae. a Zygotene c, d Diplotene showing residues of the nucleolus. e Spermatids. nuclei with nucleolus on the periphery. b Pachytene stage, the inset in the Arrowheads indicate NOR site. Scale bar = 10 μm middle shows a detail of chromosome bivalent with one large nucleolus. suitable for karyological examination due to their much longer not very likely in genus Khawia, because these tapeworms are length. This allowed the researcher to “increase the resolution characterized by a constant diploid number. of the analysis along the length of the chromosomes fourfold The number and location of NORs vary among related in comparison with the FISH on normal metaphase chromo- genomes; therefore, comparative evolutionary studies can somes” (p. 82 Zadesenets et al. 2012). After TSA-FISH, no use the position of 18S rDNA as an appropriate genetic signals localized in the interstitial position were found in mi- marker, which can be also used for the purpose of genetic totic or meiotic chromosomes of K. abbottinae. If the presence identification of related species (Singh et al. 2009; Nguyen of ITS in the species genome shows the large chromosomal et al. 2010;Silva-Netoetal.2015;Teixeiraetal.2017). rearrangements during karyotype evolution, then we could The mapping of the number and location of rDNA sites in come to conclusion that the occurrence of such sequences is Cestoda species is until now a scattered task, since only

Fig. 6 Comparison of idiograms of chromosomes of a Khawia sinensis (Orosová and Oros 2012), b K. saurogobii (Orosová et al. 2010a), c K. japonensis (Grey 1979), and d K. abbottinae (present paper), constructed from data on relative length. 1–8, chromosome pair number; red dot, location of 18S rDNA cluster 2798 Parasitol Res (2019) 118:2789–2800 four lytocestid tapeworm species had been previously Acknowledgments This work was supported by the Slovak Research and analysed. Two loci of rDNA genes per each of three chro- Development Agency under contract No. SK-CN-2017-0007 and Bilateral Mobility Project SAV-18-20 and by the Research & mosomes of the triplet 2 were revealed in the triploid par- Development Operational Program funded by the ERDF: thenogenetic species A. huronensis (Kráľová-Hromadová Environmental protection against parasitozoonoses under the influence et al. 2010; Špakulová et al. 2019). In C. fennica,FISH of global climate and social changes (code ITMS: 26220220116; rate with 18S rDNA probe identified a size variation of the two 0.2). non-homologous rDNA clusters positioned in the pericentromeric region of two metacentric chromosome Compliance with ethical standards pairs 8 and 9 (Orosová et al. 2010b). One cluster of major Conflict of interest On behalf of all authors, the corresponding author ribosomal DNA arrays was found in K. saurogobii states that there is no conflict of interest. (Orosová et al. 2010a)andK. sinensis (Orosová and Oros 2012); however, obvious interspecific differences were ob- served in rDNA location. 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